How Do Fruit Bats Infect Animals With Ebola
Emerg Infect Dis. 2013 Feb; xix(2): 270–273.
Ebola Virus Antibodies in Fruit Bats, Bangladesh
Kevin J. Olival, 
Ariful Islam, Meng Yu, Simon J. Anthony, Jonathan H. Epstein, Shahneaz Ali Khan, Salah Uddin Khan, Gary Crameri, Lin-Fa Wang, West. Ian Lipkin, Stephen P. Luby, and Peter Daszak
Abstract
To determine geographic range for Ebola virus, we tested 276 bats in Bangladesh. Five (3.five%) bats were positive for antibodies against Ebola Zaire and Reston viruses; no virus was detected by PCR. These bats might be a reservoir for Ebola or Ebola-like viruses, and extend the range of filoviruses to mainland Asia.
Keywords: bats, fruit bats, Chiroptera, Ebola virus, filovirus, viruses, Rousettus leschenaultii, natural reservoir, serology, zoonoses, Bangladesh
Filoviruses are zoonotic pathogens that cause episodic, lethal, hemorrhagic outbreaks amid humans and nonhuman primates and case-fatality rates up to fourscore% (ane). The family Filoviridae contains 2 genera: Marburgvirus, which contains Marburg virus (MARV), and Ebolavirus, which contains 4 viruses: Zaire Ebola virus (ZEBOV), Sudan Ebola virus, Reston Ebola virus (REBOV), and Côte d'Ivoire Ebola virus, and 2 tentative species (Bundibugyo Ebola virus and Lloviu Ebola virus) (2,three). Pathogenicity varies amongst Ebola viruses, from ZEBOV, which is highly lethal in humans, to REBOV, which causes disease in pigs and macaques simply asymptomatically infects humans.
Despite their role in human disease, natural reservoirs of filoviruses have remained elusive for decades. Reports advise that bats (Order Chiroptera) are the primary natural hosts, including Old Globe insectivorous bats (genera Rhinolophus and Miniopterus) and frugivorous bats (family Pteropodidae). Fruit bats of the genus Rousettus have been implicated as a reservoir of filoviruses in Africa (iv–7) and REBOV in the Philippines (viii). Lloviu Ebola virus was detected in Miniopterus schreibersii insectivorous bats from Spain and appears to cause pathologic changes in this species but is not known to infect humans (2). These studies betoken to a broad, and still poorly described, geographic distribution for viruses of the family unit Filoviridae in chiropteran hosts. We screened bats of several species from Bangladesh for Ebola virus infection to determine whether the geographic range of this virus extends to southern Asia.
The Study
We captured and sampled 276 bats (141 Rousettus leschenaultii bats, 75 Cynopterus spp. bats, 59 Megaderma lyra bats, and one Macroglossus sobrinus bat) during April 2010–March 2011 from the Faridpur, Rajbari, Lalmonirhat, and Comilla Districts in Bangladesh. All bats were identified to species in the field, except Cynopterus spp. bats, because of ambiguous multifariousness in this group; nosotros are awaiting genetic species confirmation. Bats were captured in mist nets nearly roosts or at feeding sites and were handled in accord with the Tufts Academy (Medford, MA, The states) Institutional Beast Care and Use Commission protocol (no. G2011-106).
We collected 50–800 µL of blood from brachial or cephalic veins of each bat, and diluted information technology 1:iv with phosphate-buffered saline in the field before serum was separated, as described (9). We also collected throat, urine/urogenital, and fecal swab specimens, which were placed in 750 µL of NucliSENS lysis buffer (bioMérieux, Marcy fifty'Etoile, France). All samples were collected in cryovials, placed in liquid nitrogen in the field, and maintained at −eighty°C until testing. We recorded morphologic measurements, weight, sex, age, and body condition and collected a wing biopsy specimen before releasing animals at capture sites.
We screened serum samples for IgG against REBOV and ZEBOV by using ELISA and Western blotting at the Commonwealth Scientific and Industrial Research Organisation Australian Animal Wellness Laboratory Biocontainment Facility (Geelong, Victoria, Australia). To inactivate potentially infectious agents, serum samples were heated at 56°C for 20 min before shipment. All samples were screened by using a i:1 mixture of purified recombinant nucleoproteins (0.2 mg/mL) of REBOV and ZEBOV (R + Z ELISA), which were expressed in an Escherichia coli vector that independent a histidine tag (x,11).
Potentially positive serum cutoff values were adamant to be >0.454 for the R + Z ELISA by using maximum-likelihood interpretation, gamma distribution, and 95% adventure for error (7). Potentially positive serum samples were tested past ELISA against each nucleoprotein independently to confirm reactivity and by Western blotting confronting nucleoproteins of Reston and Zaire virus strains equally described (10). Serum samples were tested at a dilution of one:50. Endpoint titrations with an optical density >3× the background reading were determined for serum samples positive confronting REBOV and ZEBOV antigens individually.
Total nucleic acids were extracted from samples (urine/urogenital, fecal, and throat swab specimens) past using the easyMAG NucliSENS platform (bioMérieux) at Columbia University (New York, NY, USA). Samples were tested for filovirus RNA (RNA polymerase factor) by using a consensus PCR protocol validated to amplify 19 diverse filovirus strains. This PCR has a sensitivity of fifty–500 RNA copies with synthetic transcripts and has been further validated with blood samples (12).
Fifteen (11%) of 141 R. lescehnaulti, 6 (8%) of 75 Cynopterus spp., and 4 (vii%) of 56 1000. lyra bats were potentially positive afterwards initial screening. V (3.v%) of 141 (95% CI 1.5%–eight.0%) R. leschenaultii bats were confirmed every bit seropositive after testing by ELISAs and Western blotting (Tabular array one). Bats were sampled during the breeding season; 21 (62%) of 34 sampled female R. leschenaultii bats were significant and 8 (23%) of 34 carried pups. We sampled three× as many males as females; all 5 confirmed virus-positive animals were healthy adult males (Tabular array 2). All 698 throat, urine/urogenital, and fecal samples were virus negative by PCR (Tabular array 2). All confirmed seropositive samples except 1 (April 2010–042) reacted more than strongly to Zaire virus antigens than Reston virus antigens (Table ane). Similarly, 2 samples (April 2010–057 and SB0311–059) showed college reactivity to ZEBOV by Western blotting, and other samples were equally reactive to REBOV.
Table 1
Ebola virus serologic assay results for bats, Bangladesh, 2010–2011*
| Year, specimen no. | Historic period of bat | Sexual practice of bat | Species or control | ELISA OD (endpoint titration) | Western absorb | ||||
|---|---|---|---|---|---|---|---|---|---|
| R + Z | R | Z | R | Z | |||||
| 2010 | |||||||||
| Rab691/d0 | ND | ND | Negative control | 0.138 | 0.116 | 0.097 | – | – | |
| Apr 2010–001 | A | F | Negative command (Rousettus leschenaultii) | 0.215 | 0.117 (50) | 0.058 (l) | – | – | |
| Apr 2010–002 | A | F | Negative control (R. leschenaultii) | 0.092 | 0.096 | 0.059 | – | – | |
| Rab691/EboV-N | ND | ND | Positive control | 2.303 | 1.72 | 1.23 | ++ | ++ | |
| Monkey/EboV | ND | ND | Positive control | 1.753 | 0.676 | 0.445 | NT | NT | |
| Apr 2010–042 | A | M | R. leschenaultii | one.512 | 0.511 (400) | 0.07 (fifty) | + | + | |
| April 2010–057 | A | M | R. leschenaultii | 0.684 | 0.072 (50) | 0.477 (800) | + | ++ | |
| 66 additional negative | ND | ND | R. leschenaultii | <0.60 | – | – | | NT | NT |
| 2011 | |||||||||
| Rab691/d0 | ND | ND | Negative control | 0.165 | 0.116 | 0.145 | – | – | |
| SB0311–115 | A | F | Negative control (Megaderma lyra) | 0.515 | 0.074 | 0.083 | – | – | |
| SB0311–117 | A | F | Negative control (M. lyra) | 0.775 | 0.075 | 0.072 | – | – | |
| Rab691/ REboV-N | ND | ND | Positive control | i.598 | 1.123 | 1.106 | ++ | ++ | |
| SB0311–001 | A | M | R. leschenaultii | 0.494 | 0.213 (50) | 0.538 (100) | + | + | |
| SB0311–004 | A | Yard | R. leschenaultii | 0.557 | 0.152 (l) | 0.497 (100) | + | + | |
| SB0311–059 | A | K | R. leschenaultii | 0.757 | 0.079 (50) | 0.816 (400) | – | ++ | |
| SB0311–016 | A | F | R. leschenaultii | 0.542 | 0.182 (100) | 0.367 (400) | NT | NT | |
| 67 additional negative | ND | ND | R. leschenaultii | <0.60 | NT | NT | NT | NT | |
| 55 boosted negative | ND | ND | M. lyra | <0.775 | NT | NT | NT | NT | |
| 75 negative | ND | ND | Cynopterus sp. | <0.595 | NT | NT | NT | NT | |
| ane negative | A | Chiliad | Macroglossus sobrinus | <0.256 | NT | NT | NT | NT | |
*Values in boldface are positive results. OD, optical density; R + Z, ELISA using a 1:1 mixture of recombinant nucleoproteins of Reston and Zaire Ebola viruses; R, Reston Ebola virus ELISA; Z, Zaire Ebola virus ELISA; ND, not determined;; A, adult; –, negative; ++, strongly positive; NT, not tested; +, positive.
Table 2
Bat specimen results for filovirus by PCR and Ebola virus by serologic analysis, Bangladesh, 2010–2011*
| Bat species, sex, and sample type | No. positive/ no. tested |
|---|---|
| Cynopterus spp., n = 75, 43 M, 32 F | |
| Carrion swab | 0/74 |
| Throat swab | 0/75 |
| Serum | 0/75 |
| Urine/urogenital swab | 0/39 |
| Macroglossus sobrinus, n = 1, ane Grand | |
| Feces swab | 0/1 |
| Throat swab | 0/1 |
| Serum | 0/1 |
| Urine/urogenital swab | 0/one |
| Megaderma lyra, n = 56, 23 M, 33 F | |
| Feces swab | 0/56 |
| Throat swab | 0/56 |
| Serum | 0/56 |
| Urine/urogenital swab | 0/50 |
| Rousettus leschenaultii, n = 141, 106 M, 34 F, 1 ND | |
| Feces swab | 0/141 |
| Throat swab | 0/140 |
| Serum | 5/141 |
| Urine/urogenital swab | 0/58 |
| Total | 5/971 |
*ND, sexual activity not determined.
Conclusions
Our study provides evidence of Ebola virus infection in wildlife from mainland Asia and corroborates the ascertainment that filoviruses are harbored across a much larger geographic range and then assumed (2). Preferential reactivity to ZEBOV suggests exposure to an Ebola virus that is distinct from REBOV, the merely filovirus currently establish in Asia. We consider the likelihood of cross-reactivity with MARV as low because there is only a 35% aa identity between nucleoprotein genes of REBOV/ZEBOV and MARV. However, we cannot rule out co-infection with multiple filoviruses.
Seroprevalence found in this study is consistent with that institute in some other study (4). However, other studies of Rousettus spp. bats have reported college values (east.g., 7%–20% and 8% of R. aegyptiacus bats seropositive for MARV and ZEBOV, respectively) (6,7), and 5 (31%) of 16 R. amplexicaudatus bats seropositive for REBOV (8). These differences might have been caused by poor specificity of the assay if this virus is novel, an artifact of low volume of claret collected, the potential that other species may have greater roles as reservoirs than Rousettus spp. in Bangladesh, or timing of sampling. R. leschenaultii bats accept a big range (China to Bharat) (xiii); and more than detailed studies of virus ecology and diversity are warranted to improve understand their part as a potential reservoir of zoonotic disease agents.
We demonstrated that serologic and virus surveys of bats can exist informative for identifying potential virus hosts. Previous studies amplified ZEBOV nucleic acid from bat feces (14). We also screened bat carrion to place potential routes of virus excretion, which is useful when the route of exposure from bats to humans is known. A short interval for Ebola virus shedding by reservoir hosts and an inverse human relationship betwixt viremia and antivirus titer probably explain our negative PCR results for seropositive bats. Failure to notice filovirus nucleic acid might reverberate our relatively minor sample size, low virus prevalence, or use of a PCR that has low sensitivity for filoviruses circulating in People's republic of bangladesh.
In Bangladesh, human outbreaks of Nipah virus take been linked to drinking date palm sap contaminated with bat excreta, presumably from Pteropus giganteus bats (fifteen). R. leschenaultii bats and other small fruit bat species visit appointment palm copse x× more than frequently than Pteropus spp. bats (15). This finding could indicate potential manual of filoviruses or whatsoever other novel viruses that R. leschenaultii bats comport. Information technology also highlights the need for more than research to empathize this ecologic organization and for amend implementation of low-cost barriers to reduce bat–human contact during periods of date palm harvesting (15).
Acknowledgments
Nosotros thank Mohammed Pitu Biswas, Mohammed Gafur Sheikh, Ausraful Islam, Najmul Haider, Mohammed Hatem Ali, Touhid Khan, Belal Uddin, Sukanta Chowdhury, and Jim Desmond for assistance in the field; Parviez Hosseini for statistical advice; and the Bangladesh Ministry of Environs and Forests for permission to conduct this study.
This written report was supported by the National Institutes of Health, the National Scientific discipline Foundation, an Environmental and Evolution of Infectious Diseases award from the Fogarty International Center (2R01-TW005869), an American Recovery and Reinvestment Act laurels (3R01TW005869-06S1), a National Plant of Allergy and Infectious Diseases Nonbiodefense Emerging Infectious Diseases Enquiry Opportunities honour (1 R01 AI079231-01) for field support, the Northeast Biodefense Heart (AI157158), the US Agency for International Development Emerging Pandemic Threats Program PREDICT Projection (Cooperative Agreement no. GHN-A-OO-09-00010-00), and the Defense Threat Reduction Agency.
Biography
•
Dr Olival is a senior research scientist at EcoHealth Alliance and former National Institutes of Health Fogarty Global Health young man. His research focuses on understanding the drivers of zoonotic disease emergence and the ecology and development of bats and their associated viruses.
Footnotes
Suggested citation for this article: Olival KJ, Islam A, Yu M, Anthony SJ, Epstein JH, Khan SA, et al. Ebola virus antibodies in fruit bats, People's republic of bangladesh. Emerg Infect Dis [Internet]. 2013 February [appointment cited]. http://dx.doi.org/10.3201/eid1902.120524
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